pepstatin a  (Millipore)

 
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    Name:
    Pepstatin A
    Description:

    Catalog Number:
    p4265
    Price:
    None
    Applications:
    Can be used in conjunction with E64-d and Leupeptin A to inhibit the degradation of autophagic cargo inside autophagosomes. For this application, the working concentration is typically between 1-10 muM.
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    Structured Review

    Millipore pepstatin a
    Pepstatin A

    https://www.bioz.com/result/pepstatin a/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pepstatin a - by Bioz Stars, 2021-03
    99/100 stars

    Images

    1) Product Images from "Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice"

    Article Title: Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice

    Journal: mBio

    doi: 10.1128/mBio.00724-15

    Effects of immune serum, HuCal I, and Pepstatin A on vaginal inflammation induced by C. albicans (A and B) and on PMN influx in mice challenged with Δ sap and control strains (C) . Vaginal washes of mice treated for 24 h with saline or CA-6 (2 × 10 7 yeast cells/10 µl/mouse) in the presence or absence of nonimmune serum, immune serum, HuCal nI, HuCal I, or Pepstatin A were analyzed for the percentage of GR-1-positive cells (A) or IL-1β production (B). (C) PMN influx (percentage of GR-1 positive cells) of vaginal washes of mice challenged with CA-6, CAI4, or Δ sap strains (2 × 10 7 yeast cells/10 µl/mouse). In all panels, data are expressed as means ± SEM. *, P
    Figure Legend Snippet: Effects of immune serum, HuCal I, and Pepstatin A on vaginal inflammation induced by C. albicans (A and B) and on PMN influx in mice challenged with Δ sap and control strains (C) . Vaginal washes of mice treated for 24 h with saline or CA-6 (2 × 10 7 yeast cells/10 µl/mouse) in the presence or absence of nonimmune serum, immune serum, HuCal nI, HuCal I, or Pepstatin A were analyzed for the percentage of GR-1-positive cells (A) or IL-1β production (B). (C) PMN influx (percentage of GR-1 positive cells) of vaginal washes of mice challenged with CA-6, CAI4, or Δ sap strains (2 × 10 7 yeast cells/10 µl/mouse). In all panels, data are expressed as means ± SEM. *, P

    Techniques Used: Mouse Assay

    Effect of immune serum, HuCal Abs, and Pepstatin A on Sap2 proinflammatory activity. Vaginal washes of mice treated for 24 h with saline, LPS (50 µg/10 µl/mouse), or Sap2 (0.5 µg/10 µl/mouse) in the presence or absence of nonimmune serum, immune serum, noninhibitory or inhibitory (HuCal nI or HuCal I) MAb, or Pepstatin A were analyzed for the percentage of GR-1-positive cells (A and C) or IL-1β production (B and D). Data are expressed as means ± SEM. *, P
    Figure Legend Snippet: Effect of immune serum, HuCal Abs, and Pepstatin A on Sap2 proinflammatory activity. Vaginal washes of mice treated for 24 h with saline, LPS (50 µg/10 µl/mouse), or Sap2 (0.5 µg/10 µl/mouse) in the presence or absence of nonimmune serum, immune serum, noninhibitory or inhibitory (HuCal nI or HuCal I) MAb, or Pepstatin A were analyzed for the percentage of GR-1-positive cells (A and C) or IL-1β production (B and D). Data are expressed as means ± SEM. *, P

    Techniques Used: Activity Assay, Mouse Assay

    Effects of immune serum, HuCal I, and Pepstatin A on vaginal colonization by C. albicans . Anesthetized mice under the pseudoestrus condition were treated intravaginally 30 min before and again 30 min after challenge with gLUC59 (2 × 10 7 yeast cells/10 µl/mouse), with rabbit nonimmune or immune serum and anti-tSap2, HuCal nI or HuCal I, Pepstatin A, or FLZ. At 2 and 5 days postchallenge, mice were treated intravaginally with 10 µl of coelenterazine (0.5 mg/ml) and imaged with the IVIS-200TM imaging system under anesthesia with 2.5% isofluorane (A). Quantification of total photon emission from ROI was evaluated, and the statistical significance was determined (B and D). #, P
    Figure Legend Snippet: Effects of immune serum, HuCal I, and Pepstatin A on vaginal colonization by C. albicans . Anesthetized mice under the pseudoestrus condition were treated intravaginally 30 min before and again 30 min after challenge with gLUC59 (2 × 10 7 yeast cells/10 µl/mouse), with rabbit nonimmune or immune serum and anti-tSap2, HuCal nI or HuCal I, Pepstatin A, or FLZ. At 2 and 5 days postchallenge, mice were treated intravaginally with 10 µl of coelenterazine (0.5 mg/ml) and imaged with the IVIS-200TM imaging system under anesthesia with 2.5% isofluorane (A). Quantification of total photon emission from ROI was evaluated, and the statistical significance was determined (B and D). #, P

    Techniques Used: Mouse Assay, Imaging

    2) Product Images from "Central Role of Mitofusin 2 in Autophagosome-Lysosome Fusion in Cardiomyocytes *"

    Article Title: Central Role of Mitofusin 2 in Autophagosome-Lysosome Fusion in Cardiomyocytes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.379164

    Defective autophagic degradation in MFN2 CKO heart. A,  increased p62 level by Western blot and statistics of the p62/tubulin ratio.  n  = 3 pairs of wild-type control and MFN2 CKO mice at 4 months old.  B,  LC3-II protein levels showing the autophagic flux determined by Western blot in Langendorff-perfused hearts under ischemia-reperfusion stress (IR) with or without the lysosomal inhibitors NH 4 Cl and pepstatin A.  C,  LC3-II/tubulin ratios from  B. n  = 3 for each group.  D,  confocal images of Langendorff-perfused hearts stained by LysoTracker TM  red before and after ischemia-reperfusion stress.  E,  surface area of LysoTracker TM  red staining counted from  D . *, significant difference between wild-type and MFN2 CKO hearts; †, significant difference between inhibitor-treated and untreated groups; #, significant difference between ischemia-reperfusion (I/R)-treated and untreated groups.
    Figure Legend Snippet: Defective autophagic degradation in MFN2 CKO heart. A, increased p62 level by Western blot and statistics of the p62/tubulin ratio. n = 3 pairs of wild-type control and MFN2 CKO mice at 4 months old. B, LC3-II protein levels showing the autophagic flux determined by Western blot in Langendorff-perfused hearts under ischemia-reperfusion stress (IR) with or without the lysosomal inhibitors NH 4 Cl and pepstatin A. C, LC3-II/tubulin ratios from B. n = 3 for each group. D, confocal images of Langendorff-perfused hearts stained by LysoTracker TM red before and after ischemia-reperfusion stress. E, surface area of LysoTracker TM red staining counted from D . *, significant difference between wild-type and MFN2 CKO hearts; †, significant difference between inhibitor-treated and untreated groups; #, significant difference between ischemia-reperfusion (I/R)-treated and untreated groups.

    Techniques Used: Western Blot, Mouse Assay, Staining

    3) Product Images from "Secreted aspartyl proteinase (PbSap) contributes to the virulence of Paracoccidioides brasiliensis infection"

    Article Title: Secreted aspartyl proteinase (PbSap) contributes to the virulence of Paracoccidioides brasiliensis infection

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006806

    Effect of PbSap inhibition in experimental PCM. Colony-forming units (A) and histological sections of lungs (B) from infected BALB/c mice treated with Pepstain A or Itraconazole (ITR) after 30 days post infection. Groups of mice included only infected (Control) or infected and treated with Pepstatin A or Itraconazole (Treated). The red arrows indicate the location of fungal cells. Data were analyzed using Student’s t-test. Error bars correspond to the standard deviation of measurements performed in triplicate, and asterisks show significant differences ( p
    Figure Legend Snippet: Effect of PbSap inhibition in experimental PCM. Colony-forming units (A) and histological sections of lungs (B) from infected BALB/c mice treated with Pepstain A or Itraconazole (ITR) after 30 days post infection. Groups of mice included only infected (Control) or infected and treated with Pepstatin A or Itraconazole (Treated). The red arrows indicate the location of fungal cells. Data were analyzed using Student’s t-test. Error bars correspond to the standard deviation of measurements performed in triplicate, and asterisks show significant differences ( p

    Techniques Used: Inhibition, Infection, Mouse Assay, Standard Deviation

    Effect of pepstatin A in interaction assay of P . brasiliensis and macrophage cells. P . brasiliensis yeast cells were treated with pepstatin A on concentrations of 15 and 30 μM during 1 h before interaction assay. Pepstatin A increase phagocytosis (A) and enhance antifungal activity of macrophages reducing colony forming units (B). Data were analyzed using Student’s t-test. Error bars correspond to the standard deviation of measurements performed in triplicate, and * indicates a significant difference (* p
    Figure Legend Snippet: Effect of pepstatin A in interaction assay of P . brasiliensis and macrophage cells. P . brasiliensis yeast cells were treated with pepstatin A on concentrations of 15 and 30 μM during 1 h before interaction assay. Pepstatin A increase phagocytosis (A) and enhance antifungal activity of macrophages reducing colony forming units (B). Data were analyzed using Student’s t-test. Error bars correspond to the standard deviation of measurements performed in triplicate, and * indicates a significant difference (* p

    Techniques Used: Activity Assay, Standard Deviation

    4) Product Images from "miR-4262 regulates chondrocyte viability, apoptosis, autophagy by targeting SIRT1 and activating PI3K/AKT/mTOR signaling pathway in rats with osteoarthritis"

    Article Title: miR-4262 regulates chondrocyte viability, apoptosis, autophagy by targeting SIRT1 and activating PI3K/AKT/mTOR signaling pathway in rats with osteoarthritis

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.5444

    TNF-α treatment significantly decreases cell viability and autophagy, as well as increases apoptosis and the expression of miR-4262 in chondrocytes. (A) The cell viability in control and TNF-α groups at 0, 12, 24, 48 h using CCK-8; (B) The cell apoptosis in control and TNF-α groups at 0, 12, 24, 48 h using TUNEL staining; (C) The expression of autophagy-related proteins, including ULK1, ALG5, Beclin-1, LC3II/LC3I, in control, TNF-α, RAPA and TNF-α + E64d + pepstatin A groups using western blotting; (D) The level of miR-4262 in control and TNF-α groups at 0, 12, 24, 48 h using quantitative reverse transcription PCR. Three independent experiments were performed in each assay. *P
    Figure Legend Snippet: TNF-α treatment significantly decreases cell viability and autophagy, as well as increases apoptosis and the expression of miR-4262 in chondrocytes. (A) The cell viability in control and TNF-α groups at 0, 12, 24, 48 h using CCK-8; (B) The cell apoptosis in control and TNF-α groups at 0, 12, 24, 48 h using TUNEL staining; (C) The expression of autophagy-related proteins, including ULK1, ALG5, Beclin-1, LC3II/LC3I, in control, TNF-α, RAPA and TNF-α + E64d + pepstatin A groups using western blotting; (D) The level of miR-4262 in control and TNF-α groups at 0, 12, 24, 48 h using quantitative reverse transcription PCR. Three independent experiments were performed in each assay. *P

    Techniques Used: Expressing, CCK-8 Assay, TUNEL Assay, Staining, Western Blot, Polymerase Chain Reaction

    Upregulated miR-4262 further decreases cell viability, autophagy, and matrix synthesis as well as increases apoptosis in TNF-α-treated chondrocytes. (A) The miR-4262 level in control, scramble (control of mimic), miR-4262 mimic, NC (control of inhibitor), and miR-4262 inhibitor groups by qPCR; (B) The cell viability in control, scramble, miR-4262 mimic, inhibitor NC, and miR-4262 inhibitor groups using CCK-8; (C) The cell apoptosis in control, scramble, miR-4262 mimic, inhibitor NC, and miR-4262 inhibitor groups using TUNEL staining; (D) The expression of autophagy-related proteins, including ULK1, ALG5, Beclin-1, LC3II/LC3I, in control, scramble, miR-4262 mimic, inhibitor NC, and miR-4262 inhibitor groups using western blotting; (E) The cell viability in control, TNF-α, TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using CCK-8; (F) The cell apoptosis in control, TNF-α, TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using TUNEL staining; (G) The expression of autophagy-related proteins, including ULK1, ALG5, Beclin-1, LC3II/LC3I, in control, TNF-α, TNF-α+E64d+pepstatin (A) TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using western blotting; (H) The expression of matrix synthesis-related proteins, such as COL2A1, ACAN, MMP-13 and ADAMTS-5, in control, TNF-α, TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using western blotting. Three independent experiments were performed in each assay. *P
    Figure Legend Snippet: Upregulated miR-4262 further decreases cell viability, autophagy, and matrix synthesis as well as increases apoptosis in TNF-α-treated chondrocytes. (A) The miR-4262 level in control, scramble (control of mimic), miR-4262 mimic, NC (control of inhibitor), and miR-4262 inhibitor groups by qPCR; (B) The cell viability in control, scramble, miR-4262 mimic, inhibitor NC, and miR-4262 inhibitor groups using CCK-8; (C) The cell apoptosis in control, scramble, miR-4262 mimic, inhibitor NC, and miR-4262 inhibitor groups using TUNEL staining; (D) The expression of autophagy-related proteins, including ULK1, ALG5, Beclin-1, LC3II/LC3I, in control, scramble, miR-4262 mimic, inhibitor NC, and miR-4262 inhibitor groups using western blotting; (E) The cell viability in control, TNF-α, TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using CCK-8; (F) The cell apoptosis in control, TNF-α, TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using TUNEL staining; (G) The expression of autophagy-related proteins, including ULK1, ALG5, Beclin-1, LC3II/LC3I, in control, TNF-α, TNF-α+E64d+pepstatin (A) TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using western blotting; (H) The expression of matrix synthesis-related proteins, such as COL2A1, ACAN, MMP-13 and ADAMTS-5, in control, TNF-α, TNF-α + scramble, TNF-α + miR-4262 mimic, TNF-α + NC, and TNF-α + miR-4262 inhibitor groups using western blotting. Three independent experiments were performed in each assay. *P

    Techniques Used: Real-time Polymerase Chain Reaction, CCK-8 Assay, TUNEL Assay, Staining, Expressing, Western Blot

    5) Product Images from "The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy"

    Article Title: The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168193

    LC3B immunostaining. A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm). B) Cal-78 and SW-1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A (Inh; striated bars) and cell viability was analysed by the MTS assay. Untreated cells were measured as controls (light grey; n = 8, mean ± S.D.).
    Figure Legend Snippet: LC3B immunostaining. A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm). B) Cal-78 and SW-1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A (Inh; striated bars) and cell viability was analysed by the MTS assay. Untreated cells were measured as controls (light grey; n = 8, mean ± S.D.).

    Techniques Used: Immunostaining, Immunolabeling, MTS Assay

    Bortezomib induces autophagy in human chondrosarcoma cells. A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC 50 values of bortezomib for 24 h. C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II. D) Quantification of the relative LC3B protein expression.
    Figure Legend Snippet: Bortezomib induces autophagy in human chondrosarcoma cells. A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC 50 values of bortezomib for 24 h. C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II. D) Quantification of the relative LC3B protein expression.

    Techniques Used: Expressing, Western Blot

    Related Articles

    Incubation:

    Article Title: Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin
    Article Snippet: To induce excitatory stress, 9 DIV rat hippocampal neurons were treated with 250 nM Veratridine (Sigma-Aldrich) in the perfusion chamber for 10 min, and YFP-Parkin recruitment was quantified. .. To inhibit lysosomal degradation of damaged mitochondria, neurons were incubated with lysosomal inhibitors—5 µM Pepstatin A (Sigma-Aldrich) and 10 µm E64D (Sigma-Aldrich)—for 3–4 h before live imaging and Antimycin treatment. ..

    Article Title: An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results
    Article Snippet: Immunoprecipitation using magnetic beadsThe anti-V5 antibody (Life Technologies #R96025) was bound to the chromatin by incubating 150 µl , 200 µl ( , and ), 250 µl ( and ) or 450 µl ( , - ) chromatin with 1 µl ( , - ) or 2 µl ( ) of the anti-V5 antibody, either overnight ( and ), for 1 hour , 2 hours ( , - ) or 4 hours ( ) in a rotating wheel at 4°C ( and ) or RT ( - ). .. For the samples with separate protease inhibitors added ( and ), in addition, also 15 µl aprotinin (Sigma-Aldrich #A6279), 0.5 µl leupeptin (Sigma-Aldrich #L2884, 1 mg/ml in MQ), 0.5 µl pepstatin (Sigma-Aldrich #P4265: 1 mg/ml in 100% Methanol) and 5 µl PMSF (Sigma-Aldrich: #P7626, 200 mM in isopropanol) were added right before the incubation. .. For each IP, either 50 µl ( , and ) or 25 µl ( , - and ) magnetic beads (Dynabeads protein G, Life Technologies #10004D) was used.

    Imaging:

    Article Title: Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin
    Article Snippet: To induce excitatory stress, 9 DIV rat hippocampal neurons were treated with 250 nM Veratridine (Sigma-Aldrich) in the perfusion chamber for 10 min, and YFP-Parkin recruitment was quantified. .. To inhibit lysosomal degradation of damaged mitochondria, neurons were incubated with lysosomal inhibitors—5 µM Pepstatin A (Sigma-Aldrich) and 10 µm E64D (Sigma-Aldrich)—for 3–4 h before live imaging and Antimycin treatment. ..

    MTT Assay:

    Article Title: Beneficial Effects of HIV Peptidase Inhibitors on Fonsecaea pedrosoi: Promising Compounds to Arrest Key Fungal Biological Processes and Virulence
    Article Snippet: Chemicals Saquinavir and nelfinavir were obtained from Hoffmann-La Roche AG (Grenzach-Wyhlen, Germany), indinavir was from Merck Sharp & Dohme GmbH (Haar, Germany) and ritonavir from Abbot Park (Illinois, USA), which were dissolved in absolute methanol to obtain a final concentration of 20 mM and stored at −20°C until use. .. Amphotericin B, itraconazole, bovine serum albumin (BSA), propidium iodide, dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye (MTT), trans -epoxysuccinyl l -leucylamido-(4-guanidino) butane (E-64), phenylmethylsulphonyl fluoride (PMSF), pepstatin A and 1,10-phenanthroline were purchased from Sigma Chemical Co. (St Louis, USA). .. Media constituents, reagents used in electrophoresis and buffer components were purchased from Amersham Life Science (Little Chalfont, UK).

    Article Title: Hypoxia inhibits TRAIL-induced tumor cell apoptosis: Involvement of lysosomal cathepsins
    Article Snippet: DEVD-AFC, IETD-AMC, DEVD-CHO and IETD-CHO were from Alexis (San Diego, CA), LEHD-CHO and LEHD-AFC from Biomol (Plymouth Meeting, PA), anti-PARP antibody from Cell Signaling Technology (Beverly, MA). .. Mouse monoclonal β -actin antibody, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin A, z-Phe-Arg-NHMec, acridine orange and MTT reduction assay kit (TOX-1 kit) were from Sigma (St. Louis, MO). .. Mouse monoclonal anti-cytochrome c IgG and anti-cathepsin L antibodies were from BD Biosciences-Pharmingen (San Diego, CA).

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    Millipore pepstatin a
    Dexa-stimulated downregulation of cFLIP L protein occurs largely independent of the proteasome, lysosomal enzymes and caspases ( A ) Molt-4 and Reh cells were treated with 30 nM Bortezomib to block proteasomal activity, after one hour cells were treated with 10 ng/ml CHX for indicated time points. Expression of cFLIP L and Noxa was analyzed by Western blotting. β-Actin served as loading control. ( B ) Molt-4 and Reh cells were treated with 30 nM Bortezomib (Bort) to block proteasomal activity, after one hour cells were treated with 300 µM Dexa for four hours. Expression of cFLIP L was analyzed by Western blotting. GAPDH served as loading control. ( C ) Proteasome activity was analyzed using Chemicon 20S Proteasome Activity Assay. Fold change of proteasome activity of two independent experiments performed in duplicates is shown. EtOH was used as solvent for Dexa. ( D ) Reh cells were incubated with inhibitors of lysosomal enzymes (10 µg/ml E64d, 10 µg/ml CA-074 methyl ester (CA), 10 µg/ml <t>Pepstatin</t> A (Pep A), 50 nM Bafilomycin A (Baf), 25 µM Chloroquine (CQ)) for one hour, followed by treatment with 300 µM Dexa for four hours. cFLIP L protein expression was analyzed using Western blotting. β-Actin served as loading control. ( E ) Jurkat and Reh cells were treated with 20 µM zVAD.fmk and 300 µM Dexa for four hours or indicated time points. cFLIP L expression was analyzed by Western blotting. GAPDH served as loading control.
    Pepstatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pepstatin a/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pepstatin a - by Bioz Stars, 2021-03
    99/100 stars
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    Dexa-stimulated downregulation of cFLIP L protein occurs largely independent of the proteasome, lysosomal enzymes and caspases ( A ) Molt-4 and Reh cells were treated with 30 nM Bortezomib to block proteasomal activity, after one hour cells were treated with 10 ng/ml CHX for indicated time points. Expression of cFLIP L and Noxa was analyzed by Western blotting. β-Actin served as loading control. ( B ) Molt-4 and Reh cells were treated with 30 nM Bortezomib (Bort) to block proteasomal activity, after one hour cells were treated with 300 µM Dexa for four hours. Expression of cFLIP L was analyzed by Western blotting. GAPDH served as loading control. ( C ) Proteasome activity was analyzed using Chemicon 20S Proteasome Activity Assay. Fold change of proteasome activity of two independent experiments performed in duplicates is shown. EtOH was used as solvent for Dexa. ( D ) Reh cells were incubated with inhibitors of lysosomal enzymes (10 µg/ml E64d, 10 µg/ml CA-074 methyl ester (CA), 10 µg/ml Pepstatin A (Pep A), 50 nM Bafilomycin A (Baf), 25 µM Chloroquine (CQ)) for one hour, followed by treatment with 300 µM Dexa for four hours. cFLIP L protein expression was analyzed using Western blotting. β-Actin served as loading control. ( E ) Jurkat and Reh cells were treated with 20 µM zVAD.fmk and 300 µM Dexa for four hours or indicated time points. cFLIP L expression was analyzed by Western blotting. GAPDH served as loading control.

    Journal: Oncotarget

    Article Title: Regulation of the antiapoptotic protein cFLIP by the glucocorticoid Dexamethasone in ALL cells

    doi: 10.18632/oncotarget.24782

    Figure Lengend Snippet: Dexa-stimulated downregulation of cFLIP L protein occurs largely independent of the proteasome, lysosomal enzymes and caspases ( A ) Molt-4 and Reh cells were treated with 30 nM Bortezomib to block proteasomal activity, after one hour cells were treated with 10 ng/ml CHX for indicated time points. Expression of cFLIP L and Noxa was analyzed by Western blotting. β-Actin served as loading control. ( B ) Molt-4 and Reh cells were treated with 30 nM Bortezomib (Bort) to block proteasomal activity, after one hour cells were treated with 300 µM Dexa for four hours. Expression of cFLIP L was analyzed by Western blotting. GAPDH served as loading control. ( C ) Proteasome activity was analyzed using Chemicon 20S Proteasome Activity Assay. Fold change of proteasome activity of two independent experiments performed in duplicates is shown. EtOH was used as solvent for Dexa. ( D ) Reh cells were incubated with inhibitors of lysosomal enzymes (10 µg/ml E64d, 10 µg/ml CA-074 methyl ester (CA), 10 µg/ml Pepstatin A (Pep A), 50 nM Bafilomycin A (Baf), 25 µM Chloroquine (CQ)) for one hour, followed by treatment with 300 µM Dexa for four hours. cFLIP L protein expression was analyzed using Western blotting. β-Actin served as loading control. ( E ) Jurkat and Reh cells were treated with 20 µM zVAD.fmk and 300 µM Dexa for four hours or indicated time points. cFLIP L expression was analyzed by Western blotting. GAPDH served as loading control.

    Article Snippet: The glucocorticoid Dexa was purchased from Sigma-Aldrich (Steinheim, Germany), the caspase inhibitor zVAD.fmk from Bachem (Heidelberg, Germany), the proteasome inhibitor Bortezomib from Selleckchem (Houston, TX, USA) and cycloheximide (CHX), E64d, CA-074 methyl ester, chloroquine, pepstatin A and Bafilomycin A from Sigma-Aldrich.

    Techniques: Blocking Assay, Activity Assay, Expressing, Western Blot, Incubation

    pUL38 inhibits cathepsin leakage-mediated cell death. (A) MRC5 cells were infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell samples were collected 72 h later. Cytosolic proteins were separated and analyzed by immunoblotting with the indicated antibodies. (B) MRC5 cells were treated with Tiron (200 μM), CPX (0.5 μM), or DMSO and then infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell samples were collected 72 h later. Cytosolic proteins were separated and analyzed by immunoblotting with the indicated antibodies. (C) MRC5 cells were treated with pepstatin A (pep A) (25 μM) plus E64d (10 μM) or with DMSO and then infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell viability was assessed at the indicated time points by CellTiter-Glo luminescent cell viability assay. The viability of cells infected with wild-type HCMV infected cells was set as 100%, and the viability of cells infected with pUL38-deficient HCMV was normalized to that of wild-type-HCMV-infected cells. Data shown represent the mean ± SD from three independent experiments. ***, P

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism

    doi: 10.1128/JVI.00191-18

    Figure Lengend Snippet: pUL38 inhibits cathepsin leakage-mediated cell death. (A) MRC5 cells were infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell samples were collected 72 h later. Cytosolic proteins were separated and analyzed by immunoblotting with the indicated antibodies. (B) MRC5 cells were treated with Tiron (200 μM), CPX (0.5 μM), or DMSO and then infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell samples were collected 72 h later. Cytosolic proteins were separated and analyzed by immunoblotting with the indicated antibodies. (C) MRC5 cells were treated with pepstatin A (pep A) (25 μM) plus E64d (10 μM) or with DMSO and then infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell viability was assessed at the indicated time points by CellTiter-Glo luminescent cell viability assay. The viability of cells infected with wild-type HCMV infected cells was set as 100%, and the viability of cells infected with pUL38-deficient HCMV was normalized to that of wild-type-HCMV-infected cells. Data shown represent the mean ± SD from three independent experiments. ***, P

    Article Snippet: The iron chelators Tiron and ciclopirox olamine (CPX), the ROS scavengers Trolox and N -acetyl- l -cysteine (NAC), the ER stress inducers tunicamycin (TM) and thapsigargin (TG), the cathepsin inhibitors pepstatin A and E64d, and the eukaryote translation inhibitor cycloheximide (CHX) were purchased from Sigma-Aldrich.

    Techniques: Infection, Cell Viability Assay

    Cells were treated with 0, 5, 10 or 20 μM lycopene (a) with or without 10 μg/mL E64d and 10 μg/mL pepstatin (b) for 24 hours. Western blotting analysis was performed using a primary antibody against LC3A/B. GAPDH served as a loading control. The LC3-II / LC3-I and LC3-II / GAPDH ratios were calculated. Cells were treated with or without 10 μM lycopene in the presence or absence of 10 μg/mL E64d and 10 μg/mL pepstatin for 24 hours (c and d). Cells stained by AO (c) or transfected with GFP-LC3B (d) were visualized using a laser scanning confocal microscope. The means of the red/green fluorescence ratios (c) or the GFP-LC3 puncta (d) for individual cells were determined for statistical analysis. The data are shown as the means ± SD of three independent experiments, and representative figures are shown. Bars = 5 μm. *: p

    Journal: Journal of Cancer

    Article Title: Lycopene upregulates ZO-1 and downregulates claudin-1 through autophagy inhibition in the human cutaneous squamous cell carcinoma cell line COLO-16

    doi: 10.7150/jca.26578

    Figure Lengend Snippet: Cells were treated with 0, 5, 10 or 20 μM lycopene (a) with or without 10 μg/mL E64d and 10 μg/mL pepstatin (b) for 24 hours. Western blotting analysis was performed using a primary antibody against LC3A/B. GAPDH served as a loading control. The LC3-II / LC3-I and LC3-II / GAPDH ratios were calculated. Cells were treated with or without 10 μM lycopene in the presence or absence of 10 μg/mL E64d and 10 μg/mL pepstatin for 24 hours (c and d). Cells stained by AO (c) or transfected with GFP-LC3B (d) were visualized using a laser scanning confocal microscope. The means of the red/green fluorescence ratios (c) or the GFP-LC3 puncta (d) for individual cells were determined for statistical analysis. The data are shown as the means ± SD of three independent experiments, and representative figures are shown. Bars = 5 μm. *: p

    Article Snippet: Reagents and antibodies The compounds used in this study included lycopene (sc-205738, Santa Cruz Biotechnology, Dallas, TX, USA) dissolved in tetrahydrofuran (0.1% was used in the non-treatment cells as solvent control), rapamycin, trehalose, E64d, pepstatin, acridine orange (AO), and dimethyl sulfoxide (DMSO) (all from Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Western Blot, Staining, Transfection, Microscopy, Fluorescence

    Cells were treated with 0, 5, 10 or 20 μM lycopene (a), and then the protein levels and phosphorylation of MTOR, p70 S6 kinase, ribosomal protein S6 and 4E-BP1 were determined by western blotting. Cells were treated with or without 10 μM lycopene in the presence or absence of 80 nM rapamycin (b and c), 1 μM torin 1 (d) or 100 mM trehalose (f) for 24 hours, and the protein levels and phosphorylation of MTOR (b, d and f) and ribosomal protein S6 (b), as well as the protein levels of ZO-1, claudin-1 (c, d and f) and LC3 (d and f), were determined by western blotting. In addition, COLO-16 cells were treated with or without 10 μM lycopene in the presence or absence of 10 μg/mL E64d and 10 μg/mL pepstatin or 80 nM rapamycin (e), and the levels of LC3 protein were determined by western blotting. GAPDH served as a loading control. The LC3-II / LC3-I or LC3-II / GAPDH ratios were calculated. The data are shown as the means ± SD of three independent experiments, and representative figures are shown. *: p

    Journal: Journal of Cancer

    Article Title: Lycopene upregulates ZO-1 and downregulates claudin-1 through autophagy inhibition in the human cutaneous squamous cell carcinoma cell line COLO-16

    doi: 10.7150/jca.26578

    Figure Lengend Snippet: Cells were treated with 0, 5, 10 or 20 μM lycopene (a), and then the protein levels and phosphorylation of MTOR, p70 S6 kinase, ribosomal protein S6 and 4E-BP1 were determined by western blotting. Cells were treated with or without 10 μM lycopene in the presence or absence of 80 nM rapamycin (b and c), 1 μM torin 1 (d) or 100 mM trehalose (f) for 24 hours, and the protein levels and phosphorylation of MTOR (b, d and f) and ribosomal protein S6 (b), as well as the protein levels of ZO-1, claudin-1 (c, d and f) and LC3 (d and f), were determined by western blotting. In addition, COLO-16 cells were treated with or without 10 μM lycopene in the presence or absence of 10 μg/mL E64d and 10 μg/mL pepstatin or 80 nM rapamycin (e), and the levels of LC3 protein were determined by western blotting. GAPDH served as a loading control. The LC3-II / LC3-I or LC3-II / GAPDH ratios were calculated. The data are shown as the means ± SD of three independent experiments, and representative figures are shown. *: p

    Article Snippet: Reagents and antibodies The compounds used in this study included lycopene (sc-205738, Santa Cruz Biotechnology, Dallas, TX, USA) dissolved in tetrahydrofuran (0.1% was used in the non-treatment cells as solvent control), rapamycin, trehalose, E64d, pepstatin, acridine orange (AO), and dimethyl sulfoxide (DMSO) (all from Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Western Blot

    Blockade of Akt induces accumulation of the autophagy protein LC3 around the parasite, vacuole-lysosome fusion and killing of T. gondii dependent on the autophagy proteins. A , HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy 2 h and 24 h post-challenge. B , HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge. C , RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy 2 h and 24 h post-challenge. D , mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IV followed by challenge with T. gondii -RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite. E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole. F , Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii -YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite G, H , mHEVc cells were transfected with Beclin 1 siRNA ( G ), Atg7 siRNA ( H ) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy at 24 h. I , mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii . 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge. J , Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii . Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

    doi: 10.1371/journal.ppat.1003809

    Figure Lengend Snippet: Blockade of Akt induces accumulation of the autophagy protein LC3 around the parasite, vacuole-lysosome fusion and killing of T. gondii dependent on the autophagy proteins. A , HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy 2 h and 24 h post-challenge. B , HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge. C , RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy 2 h and 24 h post-challenge. D , mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IV followed by challenge with T. gondii -RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite. E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole. F , Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii -YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite G, H , mHEVc cells were transfected with Beclin 1 siRNA ( G ), Atg7 siRNA ( H ) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy at 24 h. I , mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii . 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge. J , Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii . Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.

    Article Snippet: In certain experiments, mammalian cells were incubated with Akt inhibitor IV (1.25 µM; EMD Millipore, Billerica, MA), PI3K inhibitor (LY294002; 20 µM; Sigma-Aldrich; St. Louis, MO), EGFR inhibitor (AG1478; 1 µM; EMD Millipore), a broad spectrum ADAM inhibitor (GM6001; 10 µM; EMD Millipore) (all 1 h prior to challenge with T. gondii ), Pertussis Toxin (PTx; 100 ng/ml; EMD Millipore; 4 h prior to challenge), leupeptin (10 µM; EMD Millipore) and pepstatin (10 µM; EMD Millipore; both 1 h after challenge with T. gondii ), 3-methyl adenine (3MA; 10 mM; Sigma Chemical) and rapamycin (1 µM; EMD Milipore; both 2 h after challenge with T. gondii ) or vehicle.

    Techniques: Incubation, Light Microscopy, Transfection, Fluorescence, Microscopy, Electron Microscopy, Expressing, Infection